In a second step the enriched cells are examined by RT-PCR for tumor-associated expression
patterns. The mRNA strands are reverse transcribed into cDNA. Subsequently, several tumor-associated markers can be amplified using multiplex PCR and visualized.

The first lane shows the DNA size standard (DNA-Ladder). Samples 1 and 3 are positive for ALDH1 and sample 2 is negative. The PCR negative control (C-) and the positive control (StemCell) are shown in the last two lanes.

The first lane shows the DNA size standard (DNA-Ladder). Sample 1 and 4 are positive for GA733-2 and CEA, samples 2 is negative, and sample 3 is positive for GA733-2, CEA and EGFR. Actin is detected in samples 1 to 4. The PCR negative (C-) and positive control (C+) are shown in the last two lanes.

The first lane shows the DNA size standard (DNA-Ladder). Sample 1 is positive for GA733-2, Muc-1 and Her-2, samples 3, 4 and 5 are positive for GA733-2 and Muc-1, and sample 6 is positive for GA733-2. Sample 2 is negative. Actin is detected in samples 1 to 6.The PCR negative (C-) and positive control (C+) are shown in the last two lanes.

The first lane shows the DNA size standard (DNA-Ladder). Sample 1 is positive for EGFR, sample 2 is positive for PSMA and PSA, and sample 3 is positive for PSMA, PSA and EGFR. Sample 4 is negative. Actin is detected in samples 1 to 4. The PCR negative (C-) and positive control (C+) are shown in the last two lanes

In the first step, the CTCs in the blood are enriched (AdnaTest Select).
This is achieved using antibody-coated magnetic particles (beads). Several antibodies are used,
which bind with high specificity and affinity to the corresponding cancer cells. The enriched cells
are lysed and subsequently purified several times to extract mRNA.

AdnaTest workflow

AdnaTest BreastCancerSelect enables the immunomagnetic
enrichment of tumor cells via epithelial and tumor-associated antigens. Antibodies against
epithelial and tumor-associated antigens are conjugated to magnetic beads (Dynabeads) for
labeling of tumor cells in peripheral blood. Labeled cells are extracted by a magnetic particle
concentrator (AdnaMag-L and AdnaMag-S) and are subsequently lysed. The results of the
AdnaTest are generate within 8 hours.

Performance

AdnaTest EMT-2/StemCellSelect allows the enrichment of circulating tumor cells from peripheral blood and AdnaTest EMT-2/StemCellDetect allows for the subsequent analysis of EMT-2/StemCell-associated gene expression. The specificity of the detection is 90%. Using EMT-2 markers in spiking experiments, 20 tumor cells in 5 ml of whole blood are detected at a recovery rate of at least 70%. Using StemCell markers in spiking experiments, 10 tumor cells in 5 ml of whole blood are detected at a recovery rate of at least 70%.

Principle

The AndaTest EMT/StemCell allows molecular characterization of CTCs by combining the AdnaTest EMT-2/StemCellSelect with the AdnaTest EMT-2/StemCellDetect using the Combination of Combinations Principle (COCP). The AdnaTest EMT-2/Stem-CellSelect is a highly specific immunomagnetic cell selection system for enrichment of circulating tumor cells from peripheral blood. The AdnaTest EMT-2/StemDetect allows sensitive analysis of cancer-associated gene expression in immunomagnetically-enriched tumor cells by reverse transcription and PCR. The test requires the enrichment of CTC from 5 ml blood followed by reverse transcription cDNA synthesis using the AdnaTests EMT-2/StemcellDetect and AdnaWash (a specialized wash buffer that reduces leukocyte cross-reactions prior to the multiplex PCR assay). The kit contains primer-mix to amplify c-DNA target fragments of Twist, PI3Kα, Akt2 and a positive control (actin). Analysis of breast, colon, prostate and ovarian cancer-associated genes is also possible.

Procedure

The AdnaTest uses a two-step process (Select and Detect) to generate results within 5 hours (see figures “AdnaTest workflow”). AdnaTest EMT-2/StemCellSelect enables the immunomagnetic enrichment of tumor cells from whole blood via epithelial- and tumor-associated antigens. Antibodies against epithelial- and tumor-associated antigens are conjugated to magnetic beads for labeling of tumor cells. Labeled cells are extracted by a magnetic particle concentrator (AdnaMag-L and AdnaMag-S), lysed and mRNA is then purified from these lysates (see figures “AdnaTest Select – Immunomagnetic cell selection with multiple tumor-associated antibodies labeled to magnetic beads”).
In the second step, the isolated mRNA is transcribed into cDNA and can be amplified using multiplex PCR (see figures “AdnaTest Detect – Multiplex PCR of various cancer-associated tumor markers”). AdnaTest EMT-2/StemCellDetect contains Oligo (dT) 25-coated beads for the isolation of mRNA from the lysate of pre-enriched tumor cells. Reverse transcription results in cDNA that can subsequently be used as template for tumor cell detection and characterization by multiplex PCR. Multiplex PCR provides tumor-associated gene expression profiles for a variety of EMT tumor markers.

The Targets

TWIST
Basic helix-loop-helix (bHLH) transcription factors have been implicated in cell lineage determination and differentiation. The protein encoded by this gene is a bHLH transcription factor and shares similarity with another bHLH transcription factor, Dermo1. It is highly expressed in placental tissue and in adult mesodermally-derived tissues. Twist binds to the E-box elements of the Akt2 promoter, enhancing its transcriptional activity. It's likely that this transcription factor is related to the EMT phenomenon in cancer cells.

Pi3K/Akt
Pi3K activates the Akt1 and Akt2 Ser/Thr kinase. Activated Akt is responsible for proliferation and has anti-apoptotic function (inhibition of caspase 9 activity). There are several possibilities that can cause the Pi3K/Akt (mTOR) signal cascade to become unregulated in cancer. One possibility is the loss of PTEN activity, which is a regulator of Pi3K that leads to a permanent activation of the pathway. Since the Pi3K/Akt pathway is also activated via tyrosine kinases it may be affected in case of EGFR overexpression/mutation and HER2 overexpression. Another activator is the IGF receptor, which has been described in breast cancer to potentially shortcut ER dependency for proliferation if overexpressed.

ALDH1
Utilizing in vitro and in vivo experimental systems, it was shown (Ginestier et al 2007) that normal and cancer human mammary epithelial cells with increased aldehyde dehydrogenase 1 activity (ALDH1) have stem/progenitor properties. Overexpression of ALDH1 in tissue of primary breast tumors correlates significantly with poor prognosis. In breast carcinomas, high ALDH1 activity leads to increased self-renewal and tumors that mimic the heterogeneity of the parental tumor.

AdnaTest EMT-2/StemCellDetect contains Oligo (dT)25-coated beads for the isolation of mRNA from the lysate of pre-enriched tumor cells. Reverse transcription results in cDNA, which is subsequently used as template for tumor cell detection and characterization by multiplex PCR. The PrimerMix EMT-1 allows the amplification of three EMT-related genes and one control gene. The PrimerMix StemCell amplifies ALDH1, which is accepted as tumor stem-cell marker. Breast cancer associated gene expression can be determined using the PrimerMix BreastDetect. Taken together the different primer-mixes generate the following fragments:

PrimerMix EMT-2

Target gene

PCR fragment size (bp)

PI3Kα

551

AKT-2

309

TWIST1

201

Actin (internal PCR control)

120

PrimerMix Stem Cell

Target gene

PCR fragment size (bp)

ALDH1

161

Optionally, the analysis of breast, colon, prostate and ovarian cancer-associated genes is possible. The PrimerMix Colon Detect allows the amplification of three tumor-associated antigens and one control gene. The primers generate fragments of the following sizes:

PrimerMix Colon Detect

Target gene

PCR fragment size (bp)

GA733-2

395

CEA

231

EGFR

163

Actin (Internal PCR control)

120

The PrimerMix Breast Detect allows the amplification of three tumor-associated antigens and one control gene. The primers generate fragments of the following sizes:

PrimerMix Breast Detect

Target gene

PCR fragment size (bp)

GA733-2

395

Muc-1

299

Her-2

265

Actin (Internal PCR control)

120

The PrimerMix Prostate Detect allows the amplification of three tumor-associated antigens and one control gene. The primers generate fragments of the following sizes:

PrimerMix Prostate Detect

Target gene

PCR fragment size (bp)

PSMA

449

PSA

357

EGFR

163

Actin (Internal PCR control)

120

Importantly, the AdnaTest is an open platform that allows the profiling of additional custom selected mRNA targets for comphrehensive biomarker discovery.